In process biotechnology, purification of proteins from complex biological mixtures involves a series of complicated recovery steps, each of which can compromise the purity and yield of the desired product Fish et al. (1984) BioTech. 2:263.
Reducing the number of such unit processes and their complexity would significantly improve product purity and yield while reducing costs. Fusion based affinity separations provide a simple means of isolating target proteins from complex cell extracts by making use of highly specific interactions between fused peptides and small, easily immobilized ligands. LaVallie et al. (1995) Curr. Opin. Biotechnol. 6:501-506; and Linder et al. (1998) Biotech. Bioeng. 60:642-647. Although fusion-based affinity systems have been known for some time and used extensively in the laboratory, their limitations have precluded their wide use in large scale applications.
In the conventional technique, the DNA coding sequence of a target protein is joined to the DNA sequence of one of a number of binding proteins to form a single open reading frame. Expression results in a two-domain fusion protein that can be easily purified via the affinity of the binding domain for its immobilized ligand. The use of optimized affinity resins minimizes the nonspecific binding of contaminant proteins, ensuring that the fusion product is recovered at high purity. Following purification, the target protein is cleaved from the binding domain at the fusion joint, where the recognition of an appropriate protease has been inserted. The product stream of this purification is a relatively simple mixture consisting of the highly purified protein of interest, the cleaved binding domain, and a small amount of protease.
The potential of this technique for use in large scale pharmaceutical production is limited in part by complications arising from the addition of protease to the purified fusion protein solution. The primary limitation is nonspecific cleavage within the product protein by the protease, leading to the destruction of the desired protein. A second disadvantage is cost; as scales increase, more protease is required, dramatically increasing production costs. Finally, the addition of protease necessitates an additional purification step, and can complicate drug approval due to the highly bioactive nature of these enzymes.
A recent advance in this area has been the introduction of self-cleaving protein linkers, achieved by combining binding domains with modified self-splicing protein elements known as inteins. Discovered in 1990, inteins are naturally occurring internal interruptions in a variety of host proteins. Hirata et al. (1990) J. Biol. Chem. 265:6726-6733; Kane et al. (1990) Science 250:651-657; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; and Noren et al. (2000) Angew. Chem. Int. Ed. 39:450-466.
Following translation of the host protein-intein precursor sequence, the intein excises itself and ligates the flanking host protein segments (exteins) to form the native host protein and released intein. A major advantage of the claimed method is that the cleavage reaction can take place on the column, eliminating the need for any further purification. Additionally the cleavage reaction only affects the target protein, thus, nonspecifically bound contaminant proteins are not affected and are not released into the product stream. This strategy forms the foundation of the commercially available IMPACT-CN system (New England Biolabs, Beverly, Mass.). (FIG. 1A). Perler et al. (1994). Because the structural information required for splicing exists entirely within the inteins they can be used in a variety of applications involving intein insertion into foreign contexts. The ability to construct intein fusions to proteins of interest has broad potential application. Gimble (1998) Chemistry & Biology 5:R251-R256. One of these is affinity fusion-based protein purification, where an intein is used in conjunction with an affinity group to purify a desired protein. Chong et al. (1997b) Gene 192:271-281; and Chong et al. (1998b) Nucl. Acids Res. 26:5109-5115. Self-cleavage, rather than splicing of the intein releases the desired protein (FIG. 1B), thereby eliminating the need for protease addition and simplifying overall processing. However, this system has drawbacks. First, in the configuration where the product protein is released by N-terminal cleavage, the cleavage reaction requires the addition of thiol containing compounds that modify the C-terminus of the product protein. Native protein is recovered only after subsequent hydrolysis of the cleavage-inducing reagent. Chong et al. (1997a) J. Biol. Chem. 272:15587-15590. Second, where the product protein is released by C-terminal cleavage in the IMPACT-CN system, the reaction is accompanied by unwanted N-terminal cleavage, requiring the N-terminal fragment to be removed in an additional purification step (described in product literature). Third, the large size of the 56-kDa Saccharomyces cerevisiae intein in the IMPACT system can diminish solubility and purification efficiency. For this application to be more attractive, the intein must be altered to yield optimized controllable cleavage rather than splicing. Furthermore, the intein should be as small as possible for this strategy to be attractive for scaleup.
Recent studies have determined that large inteins are bipartite elements consisting of a protein splicing domain interrupted by an endonuclease domain. Dalgaard et al. (1997a) Nucl. Acids Res. 25:4626-4638; Duan et al. (1997) Cell 89:555-564; and Derbyshire et al. (1997a) Proc. Natl. Acad. Sci. USA 94:11466-11471. Because endonuclease activity is not required for protein splicing, mini-inteins with accurate but reduced splicing activity can be generated by deletion of this central domain. Derbyshire et al. (1997b); Chong et al (1997a); and Shingledecker et al. (1998) Gene 207:187-195. Mechanistic studies have also determined the roles of highly conserved residues near the intein/extein junctions in the splicing reaction (FIG. 1A). Chong et al. (1996) J. Biol. Chem. 271:22159-22168; Xu et al. (1996) EMBO J. 15:5146-5153; and Stoddard et al. (1998) Nat. Struct. Biol. 5:3-5. These residues include the initial Cys, Ser or Thr of the intein, which initiates splicing with an acyl shift, the conserved Cys, Ser or Thr immediately following the intein, which ligates the exteins through nucleophilic attack, and the conserved C-terminal His and Asn of the intein, which release the intein from the ligated exteins through succinimide formation. Mutation of these residues can be used to alter intein activity to yield isolated cleavage at one or both of the intein-extein junctions. Chong et al. (1998b) J. Biol. Chem. 273:10567-10577.
Despite insights into intein structure and function, modifications often resulted in unacceptably low activity, poor precursor stability, or insolubility. Derbyshire et al. (1997b); Chong et al. (1997b); Shingledecker et al. (1998); and Chong et al. (1998a).
U.S. Pat. No. 5,795,731 (the '731 patent), explicitly stated to be not by “another” as to the present inventive entity, relates to inteins as anti-microbial targets and genetic screens for intein function. Wood et al. AIChE (American Institute of Chemical Engineers) National Meeting, Nov. 17, 1997, Wood et al. ACS (American Chemistry Society) National Meeting, Aug. 22-27, 1998; and Wood et al., AIChE (American Institute of Chemical Engineers) National Meeting, November 1998, are also explicitly stated to be not by “another” as to the present inventive entity. These Abstracts and presentations failed to teach or suggest various methods and products of the invention, including, without limitation, purification by inactivation with intein in specific regions, pH-controllable intein splicing, and methods for determining critical, generalizable residues for varying intein activity. Furthermore, these references failed to provide sufficient details for one skilled in the art to make or use inteins or mutant inteins of the invention. The Wood 1997 Abstract and presentation also failed to teach or suggest pH sensitivity or ion sensitivity by inteins or mutant inteins. Thus, the '731 patent and the Wood Abstracts and presentations fail to teach or suggest the invention.
The N-terminal (acyl shift) and C-terminal (succinimide formation) cleavage activities of the intein are separable. A great deal of work has been done to examine the N-terminal cleavage reaction, primarily because it is very similar to the cleavage reaction exhibited by hedgehog signal proteins. The N-terminal cleavage takes place in two separate steps. In the first step, the peptide bond between the intein and the N-extein is converted to a thioester (or ester in some cases). In the second step, the thioester bond is cleaved by some sort of accessory molecule. In the case of IMPACT, a commercially available affinity system from New England BioLabs, Inc. (NEB) the accessory molecule is a strong nucleophile such as P-mercaptoethanol or dithiothreitol (DTT) both of which are strong reducing agents. The nucleophile cleaves the thioester bond, i.e., a chemical mediated cleavage and not an enzyme mediated cleavage. Thus, although the initial thioester formation is mediated by the intein, the actual cleavage of the product protein is a simple chemical cleavage of a thioester bond by a small nucleophilic molecule. Thus, the N-terminal cleavage reaction can not be accelerated beyond what can be achieved through the simple chemical thioester cleavage reaction (intein structure does not play a role) and enzymatic rates of cleavage can not be attained. That is, despite changes to the intein, cleavage will always be rate-limited by the thioester cleavage reaction. IMPACT cleavage only allows for N-terminal cleavage, thereby eliminating most of the solubility and expression level advantages associated with affinity fusion. A newly available IMPACT-CN system allows N- or C-terminal cleavage, but requires an additional purification step in the case of C-terminal cleavage. Both IMPACT ND IMPACT-CN rely on N-terminal cleavage as part of the protein purification process. Even the C-terminal cleavage reaction of IMPACT-CN is modulated by the thioester mediated N-terminal cleavage reaction as cleavage takes place at both ends of the intein.
More generally, information, documents and products cited herein show that inteins and uses thereof are known. However, prior to the invention, inteins, modifications thereof and uses thereof have suffered from unacceptably low activity, poor precursor stability, and/or insolubility; and, there has been a failure heretofore to teach or suggest addressing these problems by way of any one or any combination of: a genetic system that yields self-cleaving inteins; products therefrom; methods for using such products; inteins for bioseparations; purification of proteins, such as toxic proteins (e.g., toxic to host expressing such proteins) by inactivation with inteins, e.g., inteins in specific regions and/or pH-controllable intein splicing; methods for determining critical, generalizable residues for varying intein activity; and products from such methods and processes using such products, inter alia.
The technique of in vitro protein ligation in which a protein is generated with an N-terminal Cys residue and is then used to cleave the thoiester intermediate of another protein fusion has been shown. Evans et al. (1999a) J. Biol. Chem. 274:3923-3926; Mathys et al. (1999) Gene 231:1-13; and Evans et al. (1999b) J. Biol. Chem. 274:18359-18363. The result is a simple fusion protein in which the two subunits can theoretically be from different expression systems. Although this technique is unique and interesting, it has nothing to do with the purification of native peptides. More importantly, in cases, where C-terminal cleavage is used, several amino acids are added to the beginning of the product protein. The added amino acids are described as “specific” with the sequence (CGEQPTG (SEQUENCE ID NO:1)). Evans et al. (1999a). The first five of these amino acids are the native extein sequence for the intein and appear to be required for efficient cleavage although all this is not explicitly discussed. The studies either included 5 native C-extein residues (SIEQD (SEQ ID NO:2)), or another specific (CRAMG (SEQ ID NO:3) used to allow the addition of a Cys to the beginning of the product protein. Mathys et al. (1999). If the first of the 5 native amino acids following the intein is mutated to Met (MIEQD(SEQ ID NO:4)), then cleavage takes place rapidly in vivo, preventing the efficient purification of uncleaved precursor. Again it is not discussed whether native proteins can be purified using this system, and apparently was not attempted as part of this work. The pTWIN technique of using a two-intein system to make cyclic proteins was described by Evans et al. (1999b). Again, this has nothing to do with the purification of native peptides, and again all of the proteins have the CRAMG (SEQ ID NO:3) specific included to allow efficient C-terminal cleavage. Southworth et al. (1999) Biotech. 27:110-120.
It has been claimed that the intein systems can be used to purify native product proteins through isolated C-terminal cleavage. However, the publication does not support this conclusion and does not provide details of vector construction. In the examples shown, substantial in vivo cleavage has taken place before protein purification. See, Table 2. It is also likely that the proteins being purified here begin with a non-native Ser residue. This is not specified in the paper, but is instead based on a reference to a paper published in 1997, which also does not specify the junction but instead refers to a paper published in 1993, which also does not specify the junction residues. The 1993 paper mentions that a Ser is added to the beginning of the product protein to allow splicing, but it is not clear that it was retained or might have been removed for cleavage experiments.